Morphological analysis of cell distribution and network structure via gap junctions in swine corneal stroma

Because of corneal transplantation limitations, there is a need for cornea-specific regenerative medicine. The development of such regenerative medicine has been delayed because of the complex and unique structure of the corneal stroma. Few studies have explored the corneal stroma cell distribution and cell types in vivo. This study investigated regional differences in morphological characteristics and distributions of corneal keratocytes and immunocompetent cells in the corneal stroma to clarify their functions and structural characteristics. The porcine eyeballs were subjected to light microscopy, transmission electron microscopy, scanning electron microscopy, and immunofluorescence staining analyses. Corneal cells were primarily located in the limbus, rather than the center of the cornea; the long keratocyte diameter was largest on the epithelial side of the corneal limbus, while the short diameter was largest on the endothelial side of the central cornea. Moreover, there were significantly more corneal cells on the epithelial side than on the endothelial side in both the central and limbus areas. Gap junctions between cells in the corneal stroma were present on the surfaces of cytoplasmic processes. Many cytoplasmic processes were scattered throughout the corneal stroma; they were connected both vertically and horizontally, forming an intercellular network. Additionally, immunocompetent cells on the epithelial side suggested to participate in this network via gap junctions. The morphology of keratocytes and immunocompetent cells on the epithelial side suggests that they play important roles in corneal homeostasis.     

Corneal stromal sequestration and keratoconjunctivitis sicca in a horse

A 19-year-old Shetland pony presented with unilateral ocular discomfort and abnormal ocular appearance. Keratoconjunctivitis sicca, ulcerative keratitis and brown discoloration of the corneal stroma were identified on ophthalmic examination. The etiology of keratoconjunctivitis sicca was not determined in this case. For practical and financial reasons, the owners requested enucleation of the affected eye. Histopathologic examination revealed extensive loss of corneal epithelium overlying a zone of hypereosinophilic, degenerate, and necrotic corneal stroma. This well-circumscribed region of corneal stromal sequestration was surrounded by stromal vascularization, and an intense inflammatory, predominantly polymorphonuclear, cellular infiltrate. The clinical and histopathologic features of this case were considered remarkably similar to those observed in feline corneal stromal sequestration.     

Air assisted lamellar keratectomy for the corneal haze model

To standardize the corneal haze model in the resection depth and size for efficient corneal haze development, air assisted lamellar keratectomy was performed. The ex vivo porcine corneas were categorized into four groups depending on the trephined depth: 250 µm (G1), 375 µm (G2), 500 µm (G3) and 750 µm (G4). The stroma was equally ablated at the five measurement sites in all groups. Significant differences were observed between the trephined corneal depths for resection and ablated corneal thickness in G1 (p < 0.001). No significant differences were observed between the trephined corneal depth for resection and the ablated corneal thickness in G2, G3, and G4. The resection percentage was similar in all groups after microscopic imaging of corneal sections. Air assisted lamellar keratectomy (AK) and conventional keratectomy (CK) method were applied to six beagles, after which development of corneal haze was evaluated weekly until postoperative day 28. The occurrence of corneal haze in the AK group was significantly higher than that in the CK group beginning 14 days after surgery. Alpha-smooth muscle actin expression was significantly higher in the AK group (p < 0.001) than the CK group. Air assisted lamellar keratectomy was used to achieve the desired corneal thickness after resection and produce sufficient corneal haze.     

Identification of putative orthologs of clinically relevant antimicrobial peptides in the equine ocular surface and amniotic membrane

Objectives

This study aimed to define the antimicrobial peptide (AMP) expression pattern of the equine ocular surface and amniotic membrane using a targeted qPCR approach and 3'Tag-sequencing. It will serve as a reference for future studies of ocular surface innate immunity and amniotic membrane therapies.

Procedures

A targeted qPCR approach was used to investigate the presence of orthologs for three of the most highly expressed beta-defensins (DEFB1, DEFB4B, and DEFB103A) of the human ocular surface and amniotic membrane in equine corneal epithelium, conjunctiva, and amniotic membrane. 3'Tag-sequencing was performed on RNA from one sample of corneal epithelium, conjunctiva, and amniotic membrane to further characterize their AMP expression.

Results

Equine corneal epithelium, conjunctiva, and amniotic membrane expressed DEFB1, DEFB4B, and DEFB103A. DEFB103A was expressed at the highest amounts in corneal epithelium, while DEFB4B was most highly expressed in conjunctiva and amniotic membrane. 3'Tag-sequencing from all three tissues confirmed these findings and identified expression of five additional beta-defensins, 11 alpha-defensins and two cathelicidins, with the alpha-defensins showing higher normalized read counts than the beta-defensins.

Conclusions

This study identified AMP expression in the equine cornea and conjunctiva, suggesting that they play a key role in the protection of the equine eye, similar to the human ocular surface. We also determined that equine amniotic membrane expresses a substantial number of AMPs suggesting it could potentiate an antimicrobial effect as a corneal graft material. Future studies will focus on defining the antimicrobial activity of these AMPs and determining their role in microbial keratitis.     

Equine corneal stromal abscesses: An evolution in the understanding of pathogenesis and treatment during the past 30 years

The last 30 years have seen many changes in the understanding of the pathogenesis and treatment of equine corneal stromal abscesses (SAs). Stromal abscesses were previously considered an eye problem related to corneal bacterial infection, equine recurrent uveitis, corneal microtrauma and corneal foreign bodies in horses. They were more commonly diagnosed in horses living in subtropical climatic areas of the world. Therapeutic recommendations to treat equine SAs were historically nearly always a medical approach directed at bacteria and the often associated severe iridocyclitis. Today the pathogenesis of most equine SAs appears to be more often related to fungal inoculation of the anterior corneal stroma followed by posterior migration of the fungi deeper into the corneal stroma. There is also now an increased incidence of diagnosis of corneal SAs in horses living in more temperate climates. Medical and surgical treatments are now directed towards elimination of fungal and bacterial infections, reduction and replacement of diseased corneal stroma, and suppression of iridocyclitis. If the abscess and anterior uveitis do not respond satisfactorily to medical therapy, full thickness or split thickness lamellar keratectomy to remove the fungal hyphae and diseased stroma, followed by transplantation of healthy corneal allografts has a high rate of success in speeding healing and preserving sight. This paradigm shift in the ability to diagnose and institute therapy for corneal SAs in horses has evolved over the last 30 years, and is the focus of this paper.     

Effect of topical application of 2% lidocaine gel on corneal sensitivity of clinically normal equine eyes

Objective

To assess the degree and duration of corneal anaesthesia provided by topical application of a non-ophthalmic 2% lidocaine gel in horses.

Amniotic membrane transplantation for the treatment of feline corneal sequestrum: pilot study

Objectives To describe and evaluate the use of equine amniotic membrane trans‐plantation after lamellar keratectomy for the treatment of corneal sequestrum in cats. Methods Six cats (seven eyes) of various breed and ages with corneal sequestra were treated surgically with lamellar keratectomy and amniotic membrane transplantation. All the sequestra and a small piece of the amniotic membranes used for each surgery were submitted for histopathologic examination. Results Five of the seven eyes showed minimal level of scarring in the cornea and good transparency. No recurrences of the sequestra have been noted during the follow‐up period (3–9 months). One eye had necrosis of the amniotic membrane 2 weeks after the surgery. The sequestrum of this eye showed a high level of bacterial contamination on histopathology. Three months later the same cat developed a descemetocele in the area where the necrotic amniotic membrane was rejected. A second eye developed a perforation under the amniotic membrane two weeks after the surgery. The sequestrum of this eye was deep and without vascularization. Conclusion Amniotic membrane transplantation after lamellar keratectomy was a valid procedure for surgical treatment of corneal sequestrum in cats. The procedure resulted in excellent cosmesis and functional vision in five of seven eyes; although case selection is important, particularly to exclude the very deep and non‐vascularized sequestra.     

Tolerance of the rabbit cornea to an n-butyl-ester cyanoacrylate adhesive (Vetbond)

Objective An n‐butyl‐ester cyanoacrylate adhesive available for veterinary surgery (Vetbond^®, 3M) was tested in rabbits for corneal irritation. Procedures Two experimental procedures were used on 24 rabbits: injection of the adhesive into an intralamellar corneal pocket (n = 10) and application of the glue to a mid‐stromal corneal defect (n = 14). In both experiments the eyes were examined for 20 days for evidence of corneal irritation and tolerance. At the end of each experiment, histopathologic studies were performed on all corneas. Results The corneal reaction to the intrastromally injected cyanoacrylate was characterized clinically by slight edema and vascularization localized to the vicinity of the adhesive. A moderate foreign body‐type reaction was found histologically. Following application of the adhesive to a central stromal defect, the treated corneas remained totally clear and histopathologic examination showed that the healing process was not altered compared to the controls. The mean retention time of the glue patch was 14 days. Conclusions Intrastromal injection and surface application to a corneal defect of n‐butyl‐ester cyanoacrylate to a corneal defect induced only a mild inflammatory response and did not interfere with the reparative process. These findings suggest that this surgical adhesive would be acceptable for treating corneal ulcerations in animals.     

Deep lamellar endothelial keratoplasty in 10 horses

Objective  To describe and evaluate a surgical technique utilized for the therapy of deep corneal stromal abscesses (DSA) in horses. The DSA is excised and replaced with a partial thickness corneal lamellar allograft.
Methods  A retrospective clinical study describing the indications for the surgical technique utilized and the outcomes of this procedure in 10 eyes of 10 horses.
Results  Each affected eye had a discrete DSA within the posterior stroma. An initial partial thickness semicircular corneal incision was made at the limbus, followed by anterior stromal lamellar dissection over the lesion. After excision of the DSA and replacement with a larger diameter split-thickness donor button, the anterior stroma was replaced into its original position and the initial corneal incision was repaired. All of the animals that underwent deep lamellar endothelial keratoplasty (DLEK) procedure healed appropriately and with subjectively less postoperative scarring and complications than previously described surgical approaches to DSA.
Conclusions  This procedure is an effective technique for surgical removal of DSA in horses and, in most cases, results in a visual and cosmetically acceptable globe. The advantages of this technique compared to other surgical approaches to DSA are the peripheral location of the incision, shortened anesthesia times, the resultant minimal scarring and shorter healing times associated with DLEK.     

Keratoconus in a cynomolgus monkey

In a seven-year-old male cynomolgus monkey, erythema of the upper eyelid and forehead and corneal opacity, edema and conical protrusion in the eye were observed. At necropsy, ophthalmological and serological examinations revealed binocular corneal opacity and conical protrusion and a high IgE level, respectively. Thinning of the epithelium and stroma of the cornea were noted histopathologically. At the center of the corneal epithelium, the number of epithelial cells was reduced, their cytoplasm was poorer and the basal cells were flatter than at the periphery. Bowman's membrane was folded with partial loss or breakage. Collagen fibers were compacted or disarranged, and the keratocytes were increased in the stroma, with focal pyknosis or loss of the endothelium and folding of Descemet's membrane. Electron microscopical examination revealed atrophy of the corneal epithelial basal cells. This is the first report of a case of keratoconus in a cynomolgus monkey.     

A cross‐linked hyaluronan gel accelerates healing of corneal epithelial abrasion and alkali burn injuries in rabbits

Objective To evaluate the efficacy of a chemically modified and cross‐linked derivative of hyaluronan (CMHA‐SX) for treatment of corneal epithelial abrasion and standardized alkali burn injuries. Animals Twelve female New Zealand white rabbits in two groups were used. Procedures Bilateral 6‐mm diameter corneal epithelial abrasions were made in each of six rabbits in one group and 6‐mm standardized alkali burn injuries were made in the second group. A 1% CMHA‐SX formulation was applied topically four times per day in right eye of each rabbit for 1 week, and phosphate buffered saline (PBS) was placed in left (control) eye of each rabbit. The wound size was determined by staining with 1% fluorescein and photographed at the slit lamp with a digital camera at 0, 1, 2, 3 days postoperatively in the first group and 0, 1, 2, 3, 7, 12 days in the second group. Rabbit corneas were collected for histological examination on day 7 in the first group and day 12 in the second group. Results Closure of corneal wound in the abrasion model was complete in the CMHA‐SX treated eye by 48 h. The wound closure rate and thickness of the central corneal epithelium in the CMHA‐SX treated group was greater than in control eyes for both the abrasion and alkali burn injuries. Moreover, the CMHA‐SX treated cornea exhibited better epithelial and stromal organization than the untreated control cornea. Conclusions Chemically modified and cross‐linked derivative of hyaluronan improved corneal wound healing and could be useful for treating noninfectious corneal injuries.     

Comparative observation of freeze–thaw‐induced damage in pig,rabbit, and human corneal stroma

Objective Although variations exist between species with respect to outcomes after cryopreservation, little is known about the differences in the susceptibility of the corneal stroma to cryoinjury. We performed this study to investigate freeze–thaw‐induced damage in keratocytes and collagen in rabbit, pig, and human corneas. Animals studied Rabbit, pig, and human. Procedures We prepared 250‐μm‐thick anterior stroma from rabbit, pig, and human corneas after scraping off the epithelium and endothelium. Each 250‐μm‐thick corneal stroma without epithelium was placed in a 50‐mL tube, frozen with liquid N₂ for 15 min and taken out to thaw rapidly at 37 °C. This procedure of rapid freezing and thawing was repeated three times. Differences between the species with respect to cells and collagen structures were examined using hematoxylin–eosin (H&E) staining, terminal deoxynucleotidyl transferase‐mediated nick end labeling (TUNEL) assay, and transmission electron microscopy (TEM). We orthotopically transplanted the pig and rabbit corneal transplants after the triple freeze–thaw cycle into rabbit eyes and evaluated graft survival. Results On gross examination, rabbit corneas became opaque after the triple freeze–thaw procedure, while pig and human corneas remained transparent. Histologically, keratocytes were apoptotic on TUNEL assay and TEM in rabbit, pig, and human corneas. Collagen fibrils were fragmented and the arrangement of collagen fibrils was severely disturbed in rabbit corneas on H&E staining and TEM; collagen was well preserved in pig and human corneas. Rabbit corneal stroma underwent autolysis after transplantation, whereas the pig corneal stroma remained clear for 1 month. Conclusions Our study showed that rabbit corneal stroma was more susceptible to freeze–thaw injury than pig and human corneas.     

Central corneal thickness in koi fish: effects of age, sex, body length, and corneal diameter

OBJECTIVE: To establish the central corneal thickness (CCT) of normal koi fish by ultrasonic pachymetry, and its relationship to age, sex, body length and corneal diameter. METHODS: Age, sex and body length of 33 koi fish (17 male and 16 female fish) were recorded. Horizontal and vertical corneal diameters of each eye were obtained using Jameson calipers. Central corneal thickness of all eyes was measured by ultrasonic pachymetry. Intraocular pressure (IOP) by rebound tonometry was obtained for a subgroup of nine koi (18 eyes). RESULTS: Mean central corneal thickness was 325.9 microm. Central corneal thickness of female koi was greater than CCT of male fish (P < 0.01). Central corneal thickness increased with increasing age overall and within both sexes (P < 0.01). Central corneal thickness increased with increasing body length (P < 0.001). For male and female fish, CCT increased with increasing horizontal and vertical corneal diameters (P < 0.01). Mean horizontal corneal diameter (HCD) was 8.05 mm, mean vertical corneal diameter (VCD) was 7.38 mm, and HCD was consistently greater than VCD. Mean IOP of a subgroup of these koi was 4.9 mmHg by rebound tonometry. CONCLUSIONS: Koi CCT increases with increasing age, body length and corneal diameter.     

Early postnatal development of central corneal thickness in dogs

Objective To investigate the changes in corneal thickness that occur during maturation of the canine eye over the first months of life. Animals studied Dogs of two different breeds with ages ranging from 14 days to 42 weeks of age. Procedures The central corneal thickness was measured by ultrasonic pachymetry every week for the first month after eyelid opening (around 14 days) and then every month until 42 weeks of age. Segmented regression was applied to capture the two phases observed in the central corneal thickness plotted against age. Breed, eye and gender were also included in the model. Results Mean central corneal thickness (CCT) values initially decreased following eyelid opening, with the lowest point being reached at around 6 weeks of age. Then CCT gradually increased as the dogs matured. Differences between left and right eye were not significant. Breed and gender effects were significant factors in the statistical model. Conclusions Following eyelid opening there is an initial decrease in corneal thickness until approximately 6 weeks of age, which presumably mirrors maturation of corneal endothelial cell function. After 6 weeks of age the CCT increases with age until approximately 30 weeks of age after which there was only a gradual increase over the remainder of the study period. A similar pattern of changes in corneal thickness in humans has been previously recorded.